Our long-term goal is to develop novel therapies to improve bone formation and increase bone density in women and men with osteopenia or osteoporosis. Melatonin is a novel alternative to current treatments because it possesses multiple bone-protective actions by working through a stimulation of osteoblasts, an inhibition of osteoclasts and possibly through its free-radical scavenging properties to protect bone cells against excessive bone resorption. It is expected that long-term use of melatonin in people will prevent osteopenia and osteoporosis and reduce bone fractures through its effects on both osteoblasts and osteoclasts. The objective is to clarify the mechanism(s) underlying melatonin's effects on osteoblasts and osteoclasts in rodent bone and in vitro using a novel co-culture system consisting of human mesenchymal stem cells (hMSCs) and human peripheral blood monocytes (hPBMCs). The project goal is to identify the role of MEK5 and MEK1/2 on regulating melatonin-mediated osteoblastogenesis and osteoclastogenesis using models that more closely mimics what occurs in vivo. Our central hypothesis is that melatonin, through MEK1/2 and 5, will stimulate osteoblastogenesis and inhibit osteoclastogenesis by modulating osteoblast-specific markers, RUNX2, osteocalcin and FRA-1 and by regulating the ratios of osteoprotegerin and RANKL to inhibit osteoclastogenesis, respectively. We plan to objectively test our hypothesis by pursuing the following two specific aims: (1) Assess the effects of MEK5 on melatonin-mediated osteoblast and osteoclast differentiation and signaling in hMSC-hPBMC co-cultures; (2) Assess the role of melatonin and MEK1/2 and 5 on melatonin-mediated changes in osteoblast and osteoclast function, bone formation and density in vivo. These studies will not only clarify the mechanisms underlying melatonin's actions on osteoblasts, osteoclasts and bone but will also identify a novel target (MEK5) for the development of future bone-forming therapies to reduce fracture risk in susceptible populations.